Use of PCR-HRMA for Direct Detection and Identification of Dermatophytosis Agents from Clinical Samples
Authors:
R. Dobiáš 1,2; M. Kantorová 3; P. Jaworská 1; P. Hamal 2; J. Mrázek 3
Authors‘ workplace:
Oddělení bakteriologie a mykologie, Centrum klinických laboratoří, Zdravotní ústav se sídlem v Ostravě, vedoucí oddělení RNDr. Vladislav Holec
1; Ústav mikrobiologie, Lékařská fakulta Univerzity Palackého v Olomouci, přednosta ústavu prof. MUDr. Milan Kolář, Ph. D.
2; Oddělení molekulární biologie, Centrum klinických laboratoří, Zdravotní ústav se sídlem v Ostravě, vedoucí oddělení Mgr. Jakub Mrázek
3
Published in:
Čes-slov Derm, 93, 2018, No. 6, p. 259-265
Category:
Clinical and laboratory Research
Overview
The aim of the study was to test the utility of the PCR method in combination with High Resolution Melting Analysis (HRMA) for the detection and identification of dermatophytes directly from clinical skin and adnexa samples. The methodology should reduce the time between sampling and diagnosis, increase the diagnostic sensitivity, and overall improve patient care. In the study, 128 clinical samples from patients with suspected dermatomycosis were analyzed. To isolate fungal DNA from the clinical specimens, a KIT ZR Fungal/Bacterial DNA MiniPrepTM was used. Two sets of primers specific for a wide range of dermatophytes were used to detect ribosomal DNA regions. The real-time PCR High Resolution Melting Analysis (PCR-HRMA) method was used for dermatophyte species identification. The PCR detection success was 74% for both sets of primers, the PCR-HRMA method enabled dermatophyte species identification in all PCR positive cases. In contrast, only 52% of patients with dermatophytosis were culture positive. Microscopy from the sample was positive in 90% of patients with proven dermatophytosis. 90% of dermatophytes were successfully identified using microscopy, culture and PCR-HRMA. The most common dermatophyte species (Trichophyton rubrum, T. interdigitale, T. benhamiae) were reliably detected by this methodology. PCR detection of rDNA directly from clinical material using HRMA increased the number of species identificacions in the diagnosis of dermatophytosis. The combination of classical and molecular biologic examinations appears to be a suitable method for the rapid and reliable diagnosis of dermatophytosis.
Keywords:
Trichophyton benhamiae – dermatophytes – direct identification – ribosomal DNA – Trichophyton rubrum – Trichophyton interdigitale
Sources
1. AGHAMIRIAN, M. R., GHIASIAN, S. A. Dermatophytoses in outpatients attending the Dermatology Center of Avicenna Hospital in Qazvin, Iran. Mycoses, 2008, 51, p. 155–160
2. AMEEN, M. Epidemiology of superficial fungal infections. Clin Dermatol, 2010, 28, p. 197–201.
3. BERGMAN, A., HEIMER, D., KONDORI, N., ENROTH, H. Fast and specific dermatophyte detection by automated DNA extraction and real-time PCR. Clin Microbiol Infect, 2013, 19, p. 205–211.
4. BERGMANS, A. M., SCHOULS, L. M., VAN DER ENT, M., KLAASSEN, A., BOHM, N., WINTERMANS, R. G. Validation of PCR-reverse line blot, a method for rapid detection and identification of nine dermatophyte species in nail, skin and hair samples. Clin Microbiol Infect, 2008, 14, p. 778–788.
5. BERGMANS, A. M., VAN DER ENT, M., KLAASSEN, A., BOHM, N., ANDRIESSE, G. I., WINTERMANS, R. G. Evaluation of a single-tube real-time PCR for detection and identification of 11 dermatophyte species in clinical material. Clin Microbiol Infect, 2010, 16, p. 704–710.
6. ČMOKOVÁ, A., HAMAL, P., SVOBODOVÁ, L., HUBKA, V. Detekce, identifikace a typizace dermatofytů molekulárně genetickými metodami. Čes-slov Derm, 2014, 89, p. 175–186.
7. DE BAERE, T., SUMMERBELL, R., THEELEN, B., BOEKHOUT, T., VANEECHOUTTE, M. Evaluation of internal transcribed spacer 2-RFLP analysis for the identification of dermatophytes. J Med Microbiol, 2010, 59, p. 48–54.
8. DE HOOG, G. S., DUKIK, K., MONOD, M. et al. Toward a Novel Multilocus Phylogenetic Taxonomy for the Dermatophytes. Mycopathologia, 2017, 182, p. 5–31.
9. DIDEHDAR, M., KHANSARINEJAD, B., AMIR-RAJAB, N., SHOKOHI, T. Development of a high--resolution melting analysis assay for rapid and high-throughput identification of clinically important dermatophyte species. Mycoses, 2016, 59, p. 442–449.
10. ELAVARASHI, E., KINDO, A. J., KALYANI, J. Optimization of PCR-RFLP Directly from the Skin and Nails in Cases of Dermatophytosis. Targeting the ITS and the 18S Ribosomal DNA Regions. J Clin Diagn Res, 2013, 7, p. 646–651.
11. FAGGI, E., PINI, G., CAMPISI, E., BERTELLINI, C., DIFONZO, E., MANCIANTI, F. Application of PCR to distinguish common species of dermatophytes. J Clin Microbiol, 2001, 39, p. 3382–3385.
12. FAVRE, B., HOFBAUER, B., HILDERING, K. S., RYDER, N. S. Comparison of in vitro activities of 17 antifungal drugs against a panel of 20 dermatophytes by using a microdilution assay. J Clin Microbiol, 2003, 41, p. 4817–4819.
13. FORTINI, D., CIAMMARUCONI, A., DE SANTIS, R., FASANELLA, A., BATTISTI, A., D’AMELIO, R., LISTA, F., CASSONE, A., CARATTOLI, A. Optimization of high-resolution melting analysis for low-cost and rapid screening of allelic variants of Bacillus anthracis by multiple-locus variable-number tandem repeat analysis. Clin Chem, 2007, 53, p. 1377–1380.
14. HAVLICKOVA, B., CZAIKA, V. A., FRIEDRICH, M. Epidemiological trends in skin mycoses worldwide. Mycoses, 2008, 51, p. 2–15.
15. HUBKA, V., ČMOKOVÁ, A., SKOŘEPOVÁ, M. et al. Současný vývoj v taxonomii dermatofytů a doporučení pro pojmenovávání klinicky významných druhů. Čes-slov Derm, 2014, 89, p. 151–165.
16. HUBKA, V., VĚTROVSKÝ, T., DOBIÁŠOVÁ, S.et al. Molekulární epidemiologie dermatofytóz v České republice–výsledky dvouleté studie. Čes-slov Derm, 2014, 89, p. 167–174.
17. LENGEROVA, M., RACIL, Z., HRNCIROVA, K. et al. Rapid detection and identification of mucormycetes in bronchoalveolar lavage samples from immunocompromised patients with pulmonary infiltrates by use of high-resolution melt analysis. J Clin Microbiol, 2014, 52, p. 2824–2828.
18. LIN, J. H., TSENG, C. P., CHEN, Y. J. Rapid differentiation of influenza A virus subtypes and genetic screening for virus variants by high-resolution melting analysis. J Clin Microbiol, 2008, 46, p. 1090–1097.
19. MORIARTY, B., HAY, R., MORRIS-JONES, R. The diagnosis and management of tinea. BMJ, 2012, 345, p. 4380.
20. OHST, T., KUPSCH, C., GRASER, Y. Detection of common dermatophytes in clinical specimens using a simple quantitative real-time TaqMan polymerase chain reaction assay. Br J Dermatol, 2016, 174, p. 602–609.
21. SALGO, P., DANIEL, C., GUPTA, A., MOZENA, J., JOSEPH, S. Onychomycosis disease management. Medical Crossfire: debates, peer exchange and insights in medicine, 2003, 4, p. 1–17.
22. SPILIOPOULOU, A., BARTZAVALI, C., JELASTOPULU, E., ANASTASSIOU, E. D., CHRISTOFIDOU, M. Evaluation of a commercial PCR test for the diagnosis of dermatophyte nail infections. J Med Microbiol, 2015, 64, p. 25–31.
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Dermatology & STDs Paediatric dermatology & STDsArticle was published in
Czech-Slovak Dermatology
2018 Issue 6
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